Secretome of human bronchial epithelial cells in response to the fungal pathogen Aspergillus fumigatus analyzed by differential in-gel electrophoresis.

BACKGROUND: For years, the analysis of innate responses to the major mold pathogen Aspergillus fumigatus has been restricted to specialized cells, such as professional phagocytes. More recently, the contribution of the airway epithelial barrier has been assessed and studies have shown that it was able to sense and react to the Aspergillus infection, for example, by producing cytokines. METHODS: To further explore the reaction of the respiratory epithelium to the fungus, we analyzed the proteome response of a human bronchial epithelial cell line to Aspergillus infection using difference gel electrophoresis. We studied the protein pattern of BEAS-2B cell culture supernatant after interaction of the cells with Aspergillus during a 15-hour coculture. RESULTS: We found formerly unknown aspects of bronchial cell behavior during Aspergillus infection: bronchial cells are able to develop both cellular defense mechanisms (ie, thioredoxin system activation) and immune reactions (ie, lysosomal degranulation and cathepsin activation) in response to the fungal aggression. CONCLUSIONS: Bronchial epithelial cells appear to be a more important effector of antifungal defense than expected. Degranulation of lysosomal enzymes that might be responsible for both fungal growth inhibition and host cell damage suggests that inductors/inhibitors of these pathways may be potential targets of therapeutic intervention.

Toxoplasma gondii: flat-mounting of retina as a new tool for the observation of ocular infection in mice.

Ocular toxoplasmosis is the principal cause of posterior uveitis and a leading cause of blindness. Animal models are required to improve our understanding of the pathogenesis of this disease. The method currently used for the detection of retinal cysts in animals involves the observation, under a microscope, of all the sections from infected eyes. However, this method is time-consuming and lacks sensitivity. We have developed a rapid, sensitive method for observing retinal cysts in mice infected with Toxoplasma gondii. This method involves combining the flat-mounting of retina - a compromise between macroscopic observation and global analysis of this tissue - and the use of an avirulent recombinant strain of T. gondii expressing the Escherichia coli beta-galactosidase gene, visually detectable at the submacroscopic level. Single cyst unilateral infection was found in six out of 17 mice killed within 28 days of infection, whereas a bilateral infection was found in only one mouse. There was no correlation between brain cysts number and ocular infection.

DIGE enables the detection of a putative serum biomarker of fungal origin in a mouse model of invasive aspergillosis.

Invasive aspergillosis (IA) is a major threat for immunocompromised patients. Diagnostic difficulties often delay specific treatment initiation, which increases mortality. Finding new biomarkers to improve and speed accurate diagnosis is thus vital. To investigate the ability of proteomic methods for discovering new biomarkers of IA, we used a DIGE approach to perform a proteomic analysis on both bronchoalveolar lavages (BAL) and sera at different time-points of infection in a mouse model of invasive pulmonary aspergillosis. Progression of the infection was monitored using a bioluminescent strain of Aspergillus fumigatus. Sera proteins were enriched using the ProteoMiner kit (Biorad). This method allowed us to identify a fungal protein, the A. fumigatus major allergen Asp f 2, in sera of mice one day after the infection. However, this fungal protein was not detected three days after the infection. Importantly, in BAL, this work provides evidence of an in vivo complement evasion mechanism through the cleavage of C3b into three fragments during aspergillosis. Finally, our results underlining the inflammatory host response to IA in both lung and blood compartments at different times of infection may provide new insights into the pathophysiology of this disease.

Use of mass spectrometry to identify clinical Fusarium isolates.

Fusarium spp. have recently emerged as significant human pathogens. Identification of these species is important, both for epidemiological purposes and for patient management, but conventional identification based on morphological traits is hindered by major phenotypic polymorphism. In this study, 62 strains, or isolates, belonging to nine Fusarium species were subjected to both molecular identification TEF1 gene sequencing and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) analysis. Following stringent standardization, the proteomic-based method appeared to be both reproducible and robust. Mass spectral analysis by comparison with a database, built in this study, of the most frequently isolated species, including Fusarium solani, Fusarium oxysporum, Fusarium verticilloides, Fusarium proliferatum and Fusarium dimerum, correctly identified 57 strains. As expected, the four species (i.e. Fusarium chlamydosporum, Fusarium equiseti, Fusarium polyphialidicum, Fusarium sacchari) not represented in the database were not identified. Results from mass spectrometry and molecular identification agreed in five of the six cases in which results from morphological and molecular identification were not in agreement. MALDI-TOF yielded results within 1 h, making it a valuable tool for identifying clinical Fusarium isolates at the species level. Uncommon species must now be added to the database. MALDI-TOF may also prove useful for identifying other clinically important moulds.